The burden of diseases and pathologies that cannot be treated, and less so cured, remains elevated, notwithstanding the high number of discoveries that in the past decades were translated into therapies or cures, and resulted in improvements to health and quality of life of humans. Eminent among these are numerous forms of cancers, in particular metastatic forms of cancers, that are treated with chemo-radio-therapy or biological medicaments, or combinations thereof, with very limited success.
In the past two decades there have been numerous efforts to employ herpes simplex viruses (HSVs) as oncolytic agents (o-HSVs) to treat cancers and metastases. Examples are genetically engineered HSVs, which carry deletions of some of the viral genes in order to attenuate the viruses, and confer some degree of cancer specificity. These viruses, exemplified by the virus named HSV1716, carry the deletion of one or both copies of the γ134.5 gene, whose product contrasts the host defence exerted by activation of PKR (protein kinase R). The HSVs carrying the deletion of the γ134.5 gene gain their partial cancer-specificity by the fact that non-cancer cells mount an innate response against them such that viral replication is hindered; by contrast, some of the cancer cells exhibit defects in the innate response, and thus allow the Δ γ134.5 HSV to replicate, and consequently to kill the cancer cells. A weakness of these o-HSVs is that cancer cells are heterogeneous, and the Δ γ134.5 HSV can only kill the fraction of cancer cells defective in PKR response. For safety reasons and to achieve an improved cancer-specificity, in some instances the Δ γ134.5 HSVs have been engineered to carry further deletions, exemplified by deletion of the UL 39 gene encoding the large subunit of ribonucleotide reductase, deletion of ICP 47, etc. These additional deletions result in a further attenuation of the o-HSVs.
To overcome the limited oncolytic effect consequent to attenuation, the o-HSVs carrying the deletion of γ134.5 gene, or combination of deletions, have been further modified to encode for a chemokine or cytokine. The two pertinent examples are T-VEC and M032. The o-HSV initially named Onco-Vex, and later renamed T-Vec, encodes GM-CSF, which favours the recruitment and maturation of monocytes and dendritic cells, and thus augments the response of the treated patient to its own tumor. The effect is an enhancement of the clearance of tumors by the immune system of the treated patient. In a phase III clinical trial, it improved the outcome of patients carrying metastatic melanoma. The second example is M032, a Δ γ134.5 HSV engineered to encode the sequence of IL12. This virus is predicted to favour a Th1 response. Recruitment of patients affected by glioblastoma for treatment with M032 in a phase1 trial is open. The major limits of the attenuated viruses are twofold, (i) their overall decreased replication, which represents an obstacle both in vivo, and with respect to the production of virus stocks large enough to yield efficacious inocula; and (ii) and their limited cancer-specificity due to their ability to enter and be sequestered by normal cells. These two limits are expected to be detrimental for the clinical efficacy of the treatment.
One approach to overcome these limits has been the genetic engineering of o-HSVs which exhibit a highly specific tropism for the cancer cells, and are otherwise not attenuated. This approach has been defined as retargeting of HSV tropism to cancers-specific receptors. HSV enters cells by fusion of its envelope with cell membranes; these are either the plasma membrane or the membrane bounding the endocytic vesicles. In the latter case, the attachment of the virus to the cell surface is followed by uptake of the virus by the cell into endocytic vesicles, and subsequently by fusion of the virion envelope with the membrane of the endocytic vesicle. The virion envelope is the most external structure of the HSV particle; it consists of a membrane which carries a number of virus-encoded glycoproteins that are activated in a cascade fashion to promote the fusion of the HSV envelope with cell membranes. These glycoproteins are gC and gB, which mediate a first attachment of the HSV particle to cell surface heparan sulphate. Thereafter, gD interacts with at least two independent, alternative cell surface receptors, named Nectin 1 and HVEM or HVEA. The binding site of Nectin 1 or of HVEM on gD differ. The interaction of gD with one of the two alternative receptors induces conformational changes in gD, which are thought to activate the downstream glycoproteins gH/gL (which form a heterodimer) and gB, in a cascade fashion. gB executes the fusion of the virion envelope with the cell membrane.
The retargeting of HSV to cancer-specific receptors entails the genetic modifications of gD, such that it harbours heterologous sequences which encode for a specific ligand. Upon infection with the recombinant virus which encodes the chimeric gD-ligand glycoprotein, progeny virions are formed which carry in their envelope the chimeric gD-ligand glycoprotein, in place of wt-gD. The ligand interacts with a molecule specifically expressed on the selected cancer cell, or on a group of cancers, and enables entry of the recombinant o-HSV in the selected cancer cell. Examples of ligands that have been successfully used for retargeting of HSV are IL13α, uPaR, a single chain antibody to HER2 and a single chain antibody to EGFR.
Previous studies have disclosed the construction of two recombinants named R-LM113 and R-LM249, both retargeted to the HER2 cancer receptor. To achieve a high degree of cancer specificity, the interaction of gD with its natural receptors Nectin 1 and HVEM was abolished through deletions of specific portions of the gD molecule. R-LM113 carries the deletion of the mature gD sequence corresponding to AA 6-38. R-LM249 carries the deletion of the core region of mature gD, corresponding to AA 61-218. In both viruses, the deleted sequences were replaced with the sequence encoding a single chain antibody (sc-Fv) derived from trastuzumab, a monoclonal antibody to HER2.
The retargeting through modification of glycoproteins other than gD has been attempted with gC. The inserted ligands were EPO and IL13. The virus carrying the gC-EPO chimera attached to cells expressing the EPO receptors; however this attachment did not lead to infectious entry; rather, the virus was degraded, possibly because it was taken in and ended up in lysosomes; all in all this strategy did not result in a viable retargeted virus. The gC-IL13 chimera was present in a virus that carried a second copy of IL13 in the gD gene. The virus was retargeted. Inasmuch as all viable retargeted HSV carry the retargeting ligand in gD, it cannot be inferred from those studies whether the gC-IL13 contributed or not to the retargeting to the IL13 alpha2 receptor.
The retargeting through genetic modifications of HSV gB has, to the knowledge of the inventors, never been described.
The retargeting through genetic modifications of gH/gL has, to the knowledge of the inventors, never been described, or even been attempted.
Pertinent to the present invention are the previous findings of Cairns et al. (Journal of Virology, June 2003, Vol. 77, No. 12, p. 6731-6742), summarized in the following. In an attempt to understand which role gL plays in the heterodimer gH/gL, the deletion of the gL gene was introduced in the herpesvirus named pseudorabiesvirus (PrV), a swine herpesvirus with high homology to the human HSV. The ΔgL PrV was not viable, since it could not infect, hence could not replicate. Serial blind passages of this virus gave rise to a spontaneous mutant, named PrV-ΔgLPass, which carried a chimeric glycoprotein made of the gD sequence fused to the N-terminus of gH. An essentially similar chimera was subsequently engineered also with the corresponding HSV genes. It carries the sequence encoding the signal peptide of gD and the ectodomain of mature gD (aa 1-308) fused at the N-terminus of the mature gH. Examples where gH was partially deleted did not give rise to any functional molecule. The property of the gD-gH glycoprotein, in which the entire ectodomain of gD was fused to the N-terminus of gH (named chimera 22 in Cairns et al., supra) is pertinent here. Of note, in wt virus, the activation exerted by the receptor-bound gD on gH/gL necessarily occurs through intermolecular signaling. The inventors refer to it as trans-signaling, as opposed to a signaling that occurs intramolecularly, herein referred to as cis-signaling. It is a surprising discovery of the present inventors that the activation of gH can occur in cis. This is the case for constructs R-VG803 and R-VG809, in which the sc-Fv activates the gH moiety in the chimera itself. The chimera 22 by Cairns et al., supra, was employed in complementation assays. Specifically, the chimera 22 rescued infection of a gD−/− gH+/+ virus, or of a gH−/− gD+ virus. It was not tested for complementation of a double deletion gD−/− gH−/− virus. There are two key differences between the previous report and the present finding. First, in the complementation assays (Cairns et al., supra), the wt-gD in the gH−/− gD+ virus may have activated in trans the gH moiety which is part of the gD-gH chimera. Conversely, the gD moiety, which is part of the chimera, may have activated in trans the wt-gH present in the gD−/− gH+ virions. In either case, the activation can only have taken place in-trans, as concluded by Cairns et al., supra. Evidence for cis-activation of the gD-gH chimera was not provided and would not have been considered possible by the skilled person. Secondly, irrespective of the activation mechanism, in the complementing system the gH activation was mediated by gD, which has a binding site for gH, and not by a heterologous ligand. These results did not establish or suggest that heterologous sequences (sequences other than viral sequences), herein named ligand, could be introduced at the N-terminus of gH and even less so whether a heterologous ligand introduced at the N-terminus of gH could serve the function of retargeting the HSV tropism to a cellular receptor capable to bind the engineered ligand.
The present inventors have shown that this can indeed be done and that gH can be modified to retarget herpesvirus.